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(Circulation Research. 1995;77:37-42.)
© 1995 American Heart Association, Inc.

Articles

Acetylcholine-Sensitive Intracellular Ca²⁺ Store in Fresh Endothelial Cells and Evidence for Ryanodine Receptors

Xiaodong Wang, Frankie Lau, Li Li, Akiyoshi Yoshikawa, Cornelis van Breemen

From the Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Canada.

Correspondence to Dr Casey van Breemen, The University of British Columbia, Department of Pharmacology and Therapeutics, Faculty of Medicine, 2176 Health Sciences Mall, Vancouver, BC V6T 1Z3 Canada.

Abstract In a freshly isolated endothelial cell preparation from rabbit aorta, the regulation of the acetylcholine (ACh)–sensitive intracellular Ca²⁺ store and the effects of the Ca²⁺-induced Ca²⁺ release agonists ryanodine and caffeine were studied using fura 2 imaging fluorescence microscopy. ACh (10 µmol/L) caused a transient release of Ca²⁺ from an intracellular store, presumably via an inositol tris-phosphate–sensitive mechanism. This ACh response could be repeated in the presence of extracellular Ca²⁺ but was obtained only once in Ca²⁺-free bathing solution, which shows that a depleted intracellular Ca²⁺ store can be rapidly refilled from the extracellular space. Refilling can be prevented by the endoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid (10 µmol/L), implying that Ca²⁺ enters the cytoplasm before accumulation in the endoplasmic reticulum. Ionomycin (10 µmol/L) caused a large Ca²⁺ release even after the ACh-releasable store had been emptied, indicating the existence of other ACh-insensitive stores, perhaps including the mitochondria. In one third of the cells studied, ACh induced oscillations in [Ca²⁺]_i that were dependent on extracellular Ca²⁺. Also investigated were the effects of caffeine and ryanodine. In this cell preparation neither caffeine nor ryanodine induced a Ca²⁺ transient but instead slowly increased [Ca²⁺]_i. It was observed that both caffeine and ryanodine were able to slowly deplete the ACh-sensitive store. These results indicate the presence of functional ryanodine receptors in native endothelial cells and demonstrate overlap between the caffeine and agonist-sensitive Ca²⁺ stores. We also found that caffeine was able to directly inhibit the process of ACh-induced Ca²⁺ release. It is hypothesized that endothelial endoplasmic reticulum contains both inositol tris-phosphate receptors and ryanodine receptors but that the former class are more densely distributed.

Key Words: ryanodine • caffeine • Ca²⁺-induced Ca²⁺ release • acetylcholine

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